The effect of time-ordered-expression (TOE) mRNA vaccination on the expression of immune-related genes during the activation and maturation of BMDCs

The adjuvant translational control plays a pivotal role in shaping immune responses,which means that precise kinetic modulation of mRNA vaccines could help to elicit stronger immune responses.Here, we constructed lipid nanoparticles (LNPs) encapsulating the TOE mRNA(OVA-IL-12) encoding OVA (normal expression) and IL-12 (delayed expression), and also constructed LNPs encapsulating a mRNA simultaneously expressing OVA and IL-12 (OVAoIL-12), LNPs encapsulating mRNAs expressing OVA or IL-12 individually, and LNPs encapsulating equimolar mixtures of mRNAs encoding OVA and IL-12 (OVApIL-12) as control groups.we demonstrated that the TOE mRNA kept the activation of the mTORC1 pathway, as characterized by increased expression levels of crucial genes such as Tsc1, Pik3ca, and Eif4ebp1, which potentially contributed to the improved antigen expression in the cells.We also evaluated the overall transcriptome change after LNP transfection via RNA-seq and found that TOE mRNA transfection also markedly upregulated key genes associated with immune activation.Taken together,our study introduces a transformative platform for mRNA vaccine design that could overcome the limitations of conventional mRNA vaccines. By precisely controlling the time course of adjuvant translation, this new platform enhances vaccine efficacy without compromising safety, opening new avenues for the development of advanced therapeutic and prophylactic interventions.